Highly active anti-retroviral therapy (HAART) utilizes combinations of two or more anti-viral drugs targeted to reverse transcriptase and protease for the treatment of HIV-1 infection. Patients on HAART have witnessed rapid decreases in blood and tissue levels of HIV-1 RNA and overall positive outcomes. Despite the overall success of HAART, reservoirs of HIV-1 continue to replicate in patients on HAART, necessitating prolonged and perhaps lifetime use of this therapy. Negative side effects combined with problems of patient compliance and the emergence of multiple drug resistant variants of HIV-1 argue for the development of adjuvant or alternative therapies. One such approach involves gene therapy of hematopoietic cells susceptible to HIV-1 infection. The overall goal of this program is to genetically modify human cells for resistance to HIV-1 infection and replication. To this end a series of RNA based anti-HIV-1 agents have been developed and tested. These inhibitory RNAs target multiple steps in the viral life cycle as well as the cellular CCR5 co-receptor, and include decoys to Tat and Rev, aptamers which bind Rev and ribozymes which cleave HIV-1 RNAs or the CCR5 mRNA. Previous research has led to the development of Pol III expressed, chimeric RNAs that localize the inhibitory RNAs to the cytoplasm, nucleus or nucleolus. We have shown that nucleolar localization of an anti-HIV-1 ribozyme, TAR or RBE decoy provides potent inhibition of viral replication. By combining the composition and intracellular localization of this collection of antiviral RNAs, it should be possible to achieve long-term, synergistic inhibition of HIV-1 replication. Importantly, the use of combinatorial inhibitory RNA therapy in conjunction with HAART may provide additional synergy, allowing reduced dosing of HAART reagents. The overall objectives of this proposal are to test the expression patterns and anti-HIV-1 efficacy of different combinations of our chimeric, inhibitory RNAs in the context of integrated retroviral and lentiviral vectors. We will also address the potential for synergy between the inhibitory RNAs and several HAART reagents. This program is part of an interactive R01 program (Project 1) with Dr. Ramesh Akkina (Project 2) entitled "Modeling RNA-based HIV gene therapeutics in the SCID-hu mouse." The Specific Aims of this proposal are: 1) construction of retroviral and lentiviral vectors harboring multiple Pol m cassettes capable of simultaneously expressing different anti-HIV-1 RNAs; 2) testing expression levels and the anti-HIV activities of combinatorial constructs in cultured and primary cells stably transduced with retroviral and lentiviral vectors expressing combinations of Anti-HIV-1 RNAs; 3) examination of the mechanisms of action of the inhibitory RNAs; 4) examination of the potential for synergy between therapeutic RNAs and HAART agents in blocking infectious spread of HIV-1 in cell culture; 5) transduction of primary CD34+ cells with the single and combinatorial RNA expressing vectors to monitor differentiation in culture and differentiation in the SCID-hu mouse model. This aim is to be carried out collaboratively with Dr. Ramesh Akkina (Project 2).